In previous years, we have purified and sequenced HIV-1 p64 and p51 RT (reverse transcriptase) subunits, the p34 integrase, and the p10 gag-pol protease responsible for the processing of the gag and gag-pol fusion. A sensitive assay for the protease using defined oligopeptides was also developed. Further, a GAG precursor processing pathway that results in seven gag subunits was deduced. We also identified a novel de novo synthesized gag p4l species, initiating at an internal Met at position 142 of the gag ORF. Recombinant vaccinia viruses expressing the HIV-1 protease, p64 RT, p55, and p4l gag precursor proteins with or without protease were constructed. Protein expression has been confirmed by immunological procedures. The integration protein was also expressed in the vaccinia vector. A 15 kD C-terminal cleavage product of the p64 RT was found to have RNAse H activity, and the DNA corresponding to this protein has also been expressed in vaccinia. Using a vaccinia virus expression system, we demonstrated that self-assembly of p55 GAG protein is sufficient to form the framework of the nascent human immunodeficiency virus (HIV-1) particle. The particles which budded from the cells infected with a vaccinia-GAG construct were mostly spheres with a concentric ring of electron-dense material. Expression of the GAG and POL proteins resulted in mature particles containing a condensed core which assumed the nucleoid structure characteristic of lentiviruses. When the POL frame in the GAG-POL ORF was truncated at the end of the protease domain, p55 GAG processing was markedly reduced and the maturation of the resultant particles was defective.